Ribonucleotide reductase catalyzes the conversion of ribonucleoside diphosphates to deoxyribonucleoside diphosphates, a rate limiting step of DNA synthesis. Mammalian ribonucleotide reductase is composed of two subunits: M1, which is constitutively expressed throughout the cell cycle, and M2, whose expression is S-phase dependent. In the course of studying differentially expressed genes in human cancer, we isolated full length clones of the M2 subunit of ribonucleotide reductase from a human melanoma A2058 cell cDNA library. Northern blot analysis demonstrated that there are two major mRNA transcripts of human M2, with sizes of 1.6 and 3.4 kb. Sequence data showed that the two transcripts differ in the 3' untranslated region due to alternate use of polyadenylation sites. The cDNA clones have an open reading frame encoding 389 amino acids, one less codon than found in the murine protein. The cDNA-predicted human protein sequence is 91% similar to that of the mouse; however, the 5' and 3' untranslated mRNA sequences are totally dissimilar to the murine analog. Northern and slot blot analyses performed on RNAs isolated from Dukes' C and D human colorectal carcinomas showed a 1.8-fold increase in M2 mRNA expression in tumors compared to paired normal adjacent colonic tissue. The data are consistent with the hypothesis that M2 mRNA activity is increased to provide rate limiting precursors for DNA synthesis in proliferating colonic cancer cells.